Controlled platelet activation to monitor therapy of ADP antagonists

ABSTRACT

A method is provided of determining whether an individual has reduced ability to form platelet thrombi due to inhibition of platelet activation initiation, signal transduction and/or GPIIb/IIIa blockade. A blood sample is obtained from the individual being assessed. The blood sample is mixed in combination with 1) an anticoagulant; 2) sufficient buffer to maintain the pH and salt concentration of the anticoagulated blood within a range suitable for platelet aggregation; 3) a platelet GPIIb/IIIa receptor ligand immobilized on a solid surface; 4) one or more agents to enhance a signal transduction pathway and 5) a receptor activator. The combination is incubated under conditions for agglutinating particles. Platelet-mediated agglutination is assessed in the agitated mixture. The absence of agglutination indicates that the individual has a reduced ability to form platelet thrombi.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/886,155, filed Jul. 6, 2004, now U.S. Pat. No. 7,790,362, whichclaims the benefit of U.S. Ser. No. 60/485,703, filed Jul. 8, 2003. Bothapplications are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of diagnostic assays, and inparticular to the determination of platelet function activity on bloodsamples to study effects of anti-platelet compositions, and moreparticularly the dual use of a platelet activator (like ADP) and aplatelet inhibitor (like Prostaglandin E1 (PGE1)) for measurement ofplatelet function of P2Y12 inhibitors, including clopidogrel, CS-747(Sankyo), and ticlopidine with increased sensitivity.

2. Description of the Related Art

The role of platelets in mammalian physiology is extraordinarilydiverse, but their primary role is in promoting hemostasis. In manysituations, an evaluation of the ability of blood to clot is desired, aparameter that is frequently controlled by the ability of platelets toadhere and/or aggregate. Of interest, therefore, is the assessment ofthe adhesive functions of platelets. For example, questions of interestinclude whether to administer drugs that will block, or promote, clotformation, or whether to detect deficiencies in platelet function priorto surgical procedures. Also of interest is evaluating the effectivenessof a platelet inhibitor that is being tested as a new drug or is beingused as approved clinical treatment in a patient.

Platelets are known to aggregate under a variety of conditions and inthe presence of a number of different reagents. Platelet aggregation isa term used to describe the binding of platelets to one another.Platelet aggregation in vitro depends upon the ability of platelets tobind fibrinogen to their surfaces after activation by anaggregation-inducing agent such as ADP or collagen.

Platelets play a critical role in the maintenance of normal hemostasis.When exposed to a damaged blood vessel, platelets will adhere to exposedsub-endothelial matrix. Following the initial adhesion, various factorsreleased or produced at the site of injury such as thrombin, ADP andcollagen activate the platelets. Once platelets are activated, aconformational change occurs in the platelet glycoprotein GPIIb/IIIareceptor, allowing it to bind fibrinogen and/or von Willebrand factor.

It is this binding of the multivalent fibrinogen and/or von Willebrandfactor molecules by GPIIb/IIIa receptors on adjacent platelets thatresults in the recruitment of additional platelets to the site of injuryand their aggregation to form a hemostatic plug or thrombus.

In vitro platelet aggregometry is the laboratory method used to assessthe in vivo ability of platelets to form the aggregates leading to aprimary hemostatic plug. In this technique an aggregating agent such asADP or collagen is added to whole blood or platelet-rich plasma andaggregation of platelets monitored. Platelet aggregometry is adiagnostic tool that can aide in patient diagnosis and selection oftherapy. Current assays to measure platelet aggregation are expensive,time-consuming, cumbersome, and generally not suitable for a clinicalenvironment.

A rapid platelet function assay has recently been developed and isdescribed in U.S. Pat. No. 5,763,199 (Coller). The assay determinesglycoprotein (GP)IIb/IIIa receptor blockade in whole blood.Agglutination of small polymeric beads coated with a GPIIb/IIIa ligandsuch as fibrinogen results when the beads are contacted with whole bloodcontaining platelets with activated GPIIb/IIIa receptors that are notblocked. Failure to agglutinate indicates either failure of theGPIIb/IIIa receptors to become activated and/or blockade of theGPIIb/IIIa receptors. In a preferred embodiment, the addition of athrombin receptor activator results in an assay that is rapid andconvenient enough to be performed at the bedside and that results inagglutination of the small polymeric beads within a convenient, knownperiod of time if the GPIIb/IIIa receptors are not blocked. The assayincludes the ability to transfer blood to be tested from a collectioncontainer to an assay device without opening the collection container.This platelet aggregation assay can be conducted at the same time as theactivated clotting time (ACT), which is performed to assess the adequacyof heparinization.

Platelet aggregation plays a key role in the pathogenesis of thrombosisand acute coronary artery disease. Evidence suggests that significantplatelet function variability exists in the response to variousantiplatelet agents. It has also been demonstrated that aninter-individual variability in platelet aggregation exists when P2Y12antagonists such as clopidogrel are used for treatment of patients toachieve an anti-aggregation effect. The results of one studydemonstrated that at least 10% of patients receiving the drug did notachieve the expected platelet aggregation inhibition (Muller I, Besta F,Schulz C, Massberg S, Schonig A, Gawaz M; Prevalence of clopidogrelnon-responders among patients with stable angina pectoris scheduled forelective coronary stent placemen Thromb Haemost. 2003 May, 89(5):783-7).

Clopidogrel and ticlopidine are thienopyridine derivatives that inhibitplatelet aggregation. They are believed to inhibit the binding ofadenosine-5-diphosphate (ADP) to one of its receptors, the P2Y12receptor. The pharmacological activity of clopidogrel is very similar tothe pharmacological activity of ticlopidine. However, clopidogrel hasbeen shown to have fewer side-effects than ticlopidine. Based onmounting evidence of the efficacy of clopidogrel in thrombotic disease,the use of clopidogrel and other P2Y12 antagonists are likely toincrease significantly.

Since many patients with cardiovascular disease are currently taking oneof the thienopyridine agents, a method for detection of resistance to athienopyridine and assessment of the efficacy of thienopyridinetreatment would be beneficial. Thus, there is a need to develop an assaythat would provide information about aspirin and thienopyridine, e.g.,clopidogrel or ticlopidine, sensitivity and efficacy of treatment in agiven patient.

The effects of these agents on platelet function have been assessed withplatelet aggregometry using ADP, collagen or other platelet activators.However, since ADP activates at least two different receptors (P2Y1 andP2Y12 and perhaps P2X1), it has the potential for lower specificity andbackground noise. Collagen is another choice. However collagen is highlyvariable due its quaternary structure, which dramatically affects itability to activate platelets and due to the fact it is derived frombiological tissue and sensitive to minor changes in temperature and pH.Neither collagen nor ADP provide specificity to the P2Y12 receptor andtherefore by themselves are not the optimal choice for the determinationof the effects of P2Y12 inhibitors. In particular as has been shown inseveral studies, the choice of concentration of these two agonists hassignificant effect on the degree of inhibition to P2Y12 antagonists thatis measured.

Prostaglandins (PGs) belong to a ubiquitous class of chemicals known aseicosanoids. They are found in virtually every tissue in the body andhave a very wide spectrum of biological activities. Eicosanoids arederivatives of arachidonic acid, a polyunsaturated fatty acid. The termeicosanoids includes the family of prostaglandins (PGs), prostacyclin,thromboxanes, and leukotrienes. The PGs are divided in differentfamilies depending on their structure, each designated by a letter (A,E, F, G, H, or I). In addition to this letter, each individualprostaglandin carries a digit that indicates the number of double bondsin its fatty acid side chain. For example, prostaglandin E1 (PGE1)belongs to the E family and has only one double bond in its side chain.PGs plays an important role in platelet aggregation and hemostasis(blood clotting) and typically have a marked vasodilator effect.

PGE1 is the theoretical cyclooxygenase metabolite of dihomo-γ-linolenicacid (DGLA), but it is virtually undetectable in the plasma of normalhumans or other animals. Its pharmacology includes vasodilation,hypotension, and anti-platelet activities. PGE1 has been shown toinhibit platelet aggregation by increasing cyclic adenosinemonophosphate (cAMP) concentrations within platelets. A number of groupshave shown that the IC50 of PGE1 for the inhibition of ADP-induced humanplatelet aggregation is around 40 nM.

SUMMARY OF THE INVENTION

Accordingly an object of the present invention is to provide methods andkits for conducting an assay for platelet function activity on a bloodsample.

Another object of the present invention is to provide methods and kitsto assay a blood sample that has been affected by a P2Y12 antagonistmeasured by using a combination of ADP and PGE1 as the activator.

These and other objects of the present invention are achieved in amethod of determining whether an individual has reduced ability to formplatelet thrombi due to inhibition of platelet activation initiation,signal transduction and/or GPIIb/IIIa blockade. A blood sample isobtained from the individual being assessed. The blood sample is mixedin combination with 1) an anticoagulant; 2) sufficient buffer tomaintain the pH and salt concentration of the anticoagulated bloodwithin a range suitable for platelet aggregation; 3) a plateletGPIIb/IIIa receptor ligand immobilized on a solid surface; 4) one ormore agents to enhance a signal transduction pathway and 5) a receptoractivator. The combination is incubated under conditions foragglutinating particles. Platelet-mediated agglutination is assessed inthe agitated mixture. The absence of agglutination indicates that theindividual has a reduced ability to form platelet thrombi.

In another embodiment of the present invention, a kit is provided thatincludes an anticoagulant, and a buffer to maintain the pH and saltconcentration of the anticoagulated blood within a range suitable forplatelet aggregation. Also provided is a platelet GPIIb/IIIa receptorligand immobilized on a solid surface, and a receptor activator withadditional agents to enhance a signal transduction pathway.

In another embodiment of the present invention, a method is provided ofdetermining whether an individual has reduced ability to form plateletthrombi due to inhibition of platelet activation initiation, signaltransduction and/or GPIIb/IIIa blockade using controlled activation ofthe platelet. A platelet activator and one or platelet inhibitors areprovided. An alternate signal transduction pathway is produced.

In another embodiment of the present invention, a kit is provided thatincludes a platelet GPIIb/IIIa receptor ligand immobilized on a solidsurface, one or more agents to enhance a signal transduction pathway anda receptor activator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the mean response of one individual.

FIG. 2 illustrates the mean response of five individuals.

FIG. 3 illustrates the extent of platelet aggregation from a bloodsample treated with the antagonist of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In various embodiments of the present invention, a composition of ADPand PGE1 is utilized as an activator in measuring inhibition of plateletaggregation by P2Y12 antagonists such as thienopyridines in whole bloodsamples. Accordingly, the aforementioned compositions may be employed todetermine the effectiveness of anti-platelet therapy involving treatmentof patients with a thienopyridine. The above compositions may beemployed in conjunction with particles coated with a GPIIb/IIIa receptorligand and any other reagents necessary for conducting an assay for theefficacy of thienopyridines. A lyophilized reagent composition may beused that comprises the aforementioned activator composition andparticles. In one approach, a metered volume of a sample to be measuredsuch as whole blood is mechanically mixed with the lyophilized reagent.A change in light transmission is monitored and an index of plateletactivity is calculated. In one aspect a whole blood sample is combinedin a cuvette or a unitized cartridge with the aforementioned lyophilizedreagent. An apparatus may be employed for carrying out the assay. Theapparatus comprises a well for receiving the sample where the wellcontains the lyophilized reagent and other reagents for conducting theassay. The additional reagents may be various buffers and/orlyophilization stabilizers.

As mentioned above, in one aspect the present invention is directed to amethod for conducting an assay for platelet function activity on a wholeblood sample. In one embodiment, the sample is one that has beenaffected by an adenosine-5-phosphate (ADP) antagonist. For example, thesample may be from a patient undergoing treatment with by anadenosine-5-phosphate (ADP) antagonist. In the present invention acombination is provided in an assay medium where the combinationcomprises the sample and a composition of ADP and PGE1. Usually, thefinal concentration of ADP is 2 to 35 μM, preferably, 15 to 20 μM andthe final concentration of PGE1 is 2 to 30 nM, preferably 20 to 25 nM.

Also employed in the present methods is a reagent comprising particlescoated with a compound that can result in the specific agglutination ofplatelets, i.e., the agglutination of platelets by the specificinteraction between a receptor on the platelets and the compound on theparticles. Such compounds include, by way of illustration and notlimitation, antibodies to a platelet receptor and GPIIb/IIIa receptorligands, which may be a small organic molecule, polypeptide, protein,monoclonal antibody or nucleic acid that binds, complexes or interactswith GPIIb/IIIa receptors on the platelet surface. Platelet mediatedaggregation of the particles results when the GPIIb/IIIa receptors onthe surface of platelets bind, complex or otherwise interact with theGPIIb/IIIa receptor ligands on the particles. Typical GPIIb/IIIa ligandsinclude fibrinogen, monoclonal antibody 10E5 (Coller, et al., J. Clin.Invest. 72:325 (1983)), monoclonal antibody c7E3 (The EPICInvestigators, N.E. Journal of Med., 330:956 (1994)), von Willebrandfactor, fibronectin, vitronectin and other ligands that have an arginineglycine-aspartic acid (RGD) sequence or other peptides orpeptidomimetics that mimic this sequence (Cook, et al., Drugs of theFuture 19:135 (1994)). Other compounds of interest include thrombininhibitors, low molecular weight heparin, and so forth.

The particles to which the compound is attached are at least about 0.1microns and not more than about 20 microns. In one embodiment theparticles are about 0.1 microns to about 10 microns. In anotherembodiment the particles are at least about 1 micron and less than about8 microns. The particles can be virtually any shape, but are generallyspherical with uniform diameters. The particle may have any density, butpreferably of a density approximating water, generally from about 0.7 toabout 1.5 g/ml. The particles may or may not have a charge on thesurface, either positive or negative, preferably negative. The particlesare functionalized or functionalizable so as to covalently bind orattach such members at their surface, either directly or indirectly.

The particles may be solid (e.g., comprised of organic and inorganicpolymers or latex), oil droplets (e.g., hydrocarbon, fluorocarbon,silicon fluid), or vesicles (e.g., synthetic such as phospholipids ornatural such as cells and organdies). The solid particles are normallypolymers, either addition or condensation polymers, which are readilydispersible in a liquid Medium. Examples of suspendable particles arepolymeric materials such as latex, lipid bilayers, oil droplets, cellsand hydrogels. Other particle compositions include polymers, such asnitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), polysaccharides such as dextrans and modifieddextrans, etc.; either used by themselves or in conjunction with othermaterials. The solid particles can be comprised of polystyrene,polyacrylamide, homopolymers and copolymers of derivatives of acrylateand methacrylate, particularly esters and amides, silicones and thelike.

As mentioned above, the compound is coated on the particles. Usually,the compound is covalently attached to particles. Such covalentattachment can be accomplished by well-known techniques, commonlyavailable in the literature. See, for example, “Immobilized Enzymes,”Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol.Chem., 245:3059 (1970). Briefly, as mentioned above, the surface of theparticle may be polyfunctional or be capable of beingpolyfunctionalized. A wide variety of functional groups are available orcan be incorporated. Functional groups include carboxylic acids,aldehydes, amino groups, cyano groups, ethylene groups, hydroxyl groups,mercapto groups and the like. The manner of linking a wide variety ofcompounds to surfaces is well known and is amply illustrated in theliterature (see above). The attachment of the side member may bedirectly by a bond or indirectly through the intermediacy of a linkinggroup. The length of a linking group may vary widely, depending upon thenature of the side member and of the particle.

The ratio of molecules of compound to particle is controlled in theattachment of the molecules of compound to the particle. In one approachthe number of functionalized sites on the surface of the particle may becontrolled by adjusting the number of such sites introduced on thesurface of the particle. Alternatively, or in conjunction with theabove, the ratio of molecules of compound to particle may be controlledby adjusting the concentration of the compound in the reaction mediumfor the attachment. Other approaches will be suggested to one skilled inthe art in view of the above teaching.

The particle reagent employed in the present invention may be treatedwith a sufficient amount of material to block areas of adsorption on theparticles. Such materials will not affect the functioning of theparticles for their intended purpose in the present invention. Theblocking materials include proteins such as bovine serum albumin, bovinegamma globulin, etc., polysaccharides such as dextran, etc., and thelike. In another approach, which may be utilized in conjunction with theabove, particles are employed wherein the number of functionalized sitesfor attachment substantially reduce the adsorption area on the surfaceof the particles.

The particles usually comprise a label, either attached thereto orincorporated therein. The label may be any moiety that may be used forthe purpose of detection. The label is often a member of a signalproducing system. The label is capable of being detected directly orindirectly. The label can be isotopic or nonisotopic, usuallynon-isotopic, and can be a dye, fluorescent molecule, chemiluminescentmolecule, a catalyst, such as an enzyme, a polynucleotide coding for acatalyst, promoter, coenzyme, enzyme substrate, radioactive group, asmall organic molecule, amplifiable polynucleotide sequence, and soforth.

In one specific embodiment of the present invention, the particlescontain one or more dyes that absorb in the infrared. Such dyes includebacteriochlorin, bacteriochlorophytin, meropolymethine dyes,benzoannulenes, vinylogous porphorins, polymethine dyes, cyanines andmerocyanines, and the like. Specific dyes of interest areCopper(II)-tetra-tert-butyl-tetrakis(dimethylamino)-29H-31H-phthalocyanineandVanadyl-tetra-tert-butyl-tetrakis(dimethylamino)-29H-31H-phthalocyanine.The particular dye that is selected is one of convenience, availability,stability, compatibility with the particle and the like. These dyes maybe incorporated directly into the particle itself, throughpolymerization or passive adsorption. The dyes may be loadedindividually (i.e., sequentially) or in combination (i.e.,simultaneously). Alternatively, the dyes may be linked to the bead incombination with the linking component, such that they do not leach fromthe surface. Irrespective of the loading method used, the conditions aresuch that the particle surface is unaffected with respect to the abilityto agglutinate under appropriate conditions.

The dyes absorb light in the range of about 750 nm-900 nm, particularlyin the range of about 750-850 nm. For samples with high levels of redblood cells, the light is at about 800 nm±10 nm, which is the isobesticpoint for oxyhemoglobin and reduced hemoglobin. The amount of dyeemployed with the particles varies with the extinction coefficient ofthe dye in the light range of interest, the required sensitivity of theassay, the size of the particles, the mode of binding of the dye to theparticles, compatibility of the dye with the particle matrix, and thelike. Usually, the amount of dye incorporated is in the range of about 1to 20 weight percent, more usually 5 to 15 weight percent. Dyes whichfind a particular use in the present invention are phthalocyanines.Metal free phthalocyanines absorb at approximately 700 nm (e=162,000).The metal complexes shift the absorption to either shorter or longerwavelength, most metals shift the absorption to a much shorterwavelength, but some, such as lead absorb at much longer wavelength thanthe metal free phthalocyanines.

The complexes formed between transition metals and phthalocyanines(metollophthalocyanines and Metallonaphthalocyanines) are chemicallyvery stable to light and heat. They are formed by condensation ofopthalodinitriles in the presence of an appropriate metal. Some of themetals used in the formation of the metalophthalocyanines besides thecopper (Cu) and the Vanadium (V) are magnesium (Mg), zinc (Zn), andcobalt (Co).

In one specific embodiment of the invention carboxylated microparticleswith a flat absorption maximum are employed. These microparticles areprepared by incorporating multiple dyes that have distinct absorptionmaximum close to 805 nm. This results in a flat maximum absorptionspectrum across a broad range wavelength from 780-820 nm.

The sample may be any solution, synthetic or natural, to be analyzedwhere the sample has been subject to an effect from a P2Y12 antagonist,particularly, a thienopyridine, potentially in combination with aspirin.The term sample includes biological tissue, including body fluids, froma host, and so forth. The sample can be examined directly or may bepretreated, usually. The present invention has particular application tosamples that comprise platelets, including body fluids such as, forexample, whole blood, platelet-containing blood fractions such asplasma, and the like. In one embodiment the invention has particularapplication to whole blood samples. The amount of the sample depends onthe nature of the sample. For fluid samples such as whole anticoagulatedblood, the amount of the sample is usually about 30 μl to 5000 μl,preferably, about 100 to 300 μl. The term “sample” includes unprocessedsamples directly from a patient or samples that have been pretreated andprepared in any convenient liquid medium, usually an aqueous medium(e.g., sodium citrate).

Preferably, the medium for conducting the assays in accordance with thepresent invention is an aqueous medium. Other polar cosolvents may alsobe employed in the medium, usually oxygenated organic solvents of from1-6, more usually from 1-4 carbon atoms, including alcohols, ethers andthe like. Usually, such cosolvents are present in less than about 70weight percent, more usually, in less than about 30 weight percent.Additionally, various ancillary materials are frequently employed in themethod in accordance with the present invention. For example, buffersare normally present in the assay medium, as well as stabilizers for theassay medium and the assay components; surfactants, particularlynon-ionic surfactants; binding enhancers, e.g., polyalkylene glycols; orthe like.

The pH for the medium is usually in the range of about 2 to about 11,preferably, about 4 to about 9. Various buffers may be used to achievethe desired pH and maintain the pH during the method. Illustrativebuffers include HEPES, borate, phosphate, carbonate, Tris, barbital, andthe like. The particular buffer employed is not critical to the methodbut one buffer may be preferred over others in certain circumstances. Insome circumstances HEPES is preferred and is present at a concentrationof about 0.05M to about 0.001M but generally at a concentration of about0.01M.

The volume of assay medium is about 25 to about 500 microliters, usuallyabout 75 to about 250 microliters. The assays may be carried out in anysuitable container. Conveniently, the container is a cuvette orcartridge that is used with the instrument for carrying out the assayand measuring the assay results. The reaction container usually containsthe activation initiator in accordance with the present invention in drylyophilized form together with other reagents such as the particlereagent and the like, stabilizers and so forth.

The combination of sample and particle reagent is incubated underconditions for agglutinating the particles. Moderate temperatures arenormally employed for carrying out the method. The temperature may beconstant or may vary. Usually, a constant temperature is employed duringthe reaction step. The temperature employed is usually about 10 to about80° C., more usually, about 15 to about 45° C., preferably, thetemperature should be at least 25° C., more preferably in the range ofabout 30 to about 40° C., usually about 37° C.

The extent of agglutination of the particles is determined and isrelated to the presence and/or amount of the member in the sample. Thepresence of agglutination may be determined visually by observingclumping of the particles, which would indicate agglutination.Preferably, as mentioned above, the particles may be colored to aid invisualizing agglutination or clumping of the matrix. The extent ofagglutination may be measured spectrophotometrically, turbidimetrically,nephelometrically, etc., by observing the rate of change of opticaldensity of the medium, and so forth.

In a specific embodiment of the present invention an assay for plateletfunction activity is conducted on a whole blood sample from a patientundergoing treatment with a thienopyridine. The sample is combined in asuitable container, e.g., reaction cuvette, with fibrinogen coatedparticles, and the composition of ADP and PGE1 to form an assay medium.The particles of the particle reagent have one or more infrared dyesincorporated therein. The combination is subjected to agglutinationconditions. Then, the medium is irradiated with light in the infraredregion. The transmission of infrared light from the assay mixture isdetermined where the level of transmission is related to plateletfunction activity.

The agglutination medium is selected to have high absorption at ˜800 nm.The ratio between the agglutination medium absorption coefficient andwhole blood absorption coefficient should preferably be greater thanabout 4:1 at 800 nm. The absorption ratio for a particular assay is afunction of both the absorption coefficient of the agglutination mediumand the concentration of the agglutination medium in the assay sample.

After the sample has been combined with the reagents, desirably it willbe heated to a temperature above room temperature, but below that whichwould interfere with the assay, so as to insure that the temperature canbe controlled without adversely affecting the assay result. Desirably,the temperature should be at least 25°, preferably in the range of30-40° C., more preferably about 37° C. The reaction medium is usuallygently agitated upon combining of the reagents with the sample andduring the period of the reaction. Agitation is sufficient to achieveand maintain homogeneity in the assay samples. The total time of thereadings from the zero time (time of mixing), may range from about 10sec. to 10 min., more usually about 30 sec. to 8 min., and preferablyabout 30 sec. to 3 min. The data may be analyzed by any convenientmeans, particularly using an algorithm that can manipulate the data inrelation to calibrators and/or controls.

The level of agglutination is an indication of the platelet functionactivity of the sample tested. The level of agglutination may becompared against a standard of known platelet function activity.Usually, the result will be compared to a calibrator, which may beperformed concomitantly or have been performed previously or, may beprovided as a standard curve.

The method of the present invention may be employed in conjunction withan assay for platelet count such as that described in U.S. patentapplication Ser. No. 09/177,884 filed Oct. 23, 1998 (the '884application), the relevant disclosures of which are incorporated hereinby reference.

The above assays preferably may be conducted in a device, which allowsthe reactions in accordance with the present invention to occur andwhich measures the results thereof. The instrument should assessplatelet function based upon the ability of activated platelets to bindfibrinogen. As activated platelets bind and agglutinatefibrinogen-coated particles, there is an increase in lighttransmittance. In general, an instrument to measure the result of theassay is one that can measure agglutination. Preferably, the instrumentmeasures a change in optical signal due to agglutination. Suitableinstruments include, by way of illustration and not limitation a kineticspectrophotometer, Ultegra System® instrument (commercially availablefrom Accumetrics, San Diego, Calif. and employed for rapid plateletfunction activity measurements on normal samples), or the like.

The Ultegra® System instrument is a turbidometric based opticaldetection system, that measures platelet induced aggregation as anincrease in light transmittance. The system consists of an analyzer,disposable cartridge and controls. The cartridge contains reagents basedon microparticle agglutination technology. The quality control systemincludes an electronic control, two levels of assayed “wet” controls(WQC), an in-cartridge humidity sensor, an in-packaging temperatureindicator, and a test for concurrence of two assay channels. Theanalyzer controls assay sequencing, establishes the assay temperature,controls the reagent-sample mixing for the required duration, determinesthe degree of platelet function, displays the result and performsself-diagnostics. For use in the present methods the test cartridge ofthe system contains a lyophilized preparation comprising particles withcovalently attached GPIIb/IIIa receptor ligand, a composition of ADP andPGE1, and buffer. The patient sample is usually citrated whole blood,which is automatically dispensed from the blood collection tube into thecartridge by the analyzer, with no blood handling required by the user.The interaction is monitored by the infrared absorbency characteristicsof the particles. As the particles interact with the platelets, theagglutination of the particles is measured through the optical system ofthe Ultegra™ analyzer. The agglutination is detected as an increase inthe transmission of infrared light through the sample. The reactionkinetics are analyzed and translated into “P2Y12 Response Units”, PRU.

In another embodiment of the present invention is a kit that includes inpackaged combination a lyophilized preparation comprising particles withcovalently attached fibrinogen, composition of ADP and PGE1, and buffer.The lyophilized preparation may be present in a reaction container suchas a cartridge used in the instrument of analysis. For theaforementioned Ultegra® System, the lyophilized preparation may beplaced in the outer wells of the four-well cartridge used in theanalyzer. The kit may also include a sample collection container and/ora device for carrying out the present method. The relative amounts ofreagents may be varied widely to provide for concentrations in solutionof the reagents that substantially optimize the sensitivity of adetermination.

Where appropriate, the reagents can be placed in an air-tight package inorder to maintain the activity of any reagents. The package may be, forexample, a bag, pouch, or the like fabricated from a material that issubstantially non-permeable to moisture. Such materials include, by wayof example and not limitation, plastic, aluminum foil, and the like. Forblood samples the kit may also include an article for piercing aperson's skin, disinfectant or sterilizing pads and so forth. The kitmay also include calibrators and standards. Furthermore, the kit mayalso include one or more reagents for conducting an assay for plateletcount.

The kit can include the reagents necessary for carrying out the assay ofthe present invention. In one embodiment, the kit includes a blood vial,a buffer that maintains the pH and salt concentration of the bloodsample assessed within ranges suitable for platelet mediatedagglutination of the solid surface and small polymeric beads coated withplatelet GPIIb/IIIa receptor ligand. The buffer can be in solution, orcan consist solely of the buffering composition and salts to which aknown amount of water is added to give the desired buffer solution.Optionally, the kit can also comprise an anticoagulant. In oneembodiment, the buffer is HEPES; the anticoagulant is citrate; aGPIIb/IIIa receptor ligand is fibrinogen; small polymeric beads arepolyacrylonitrile or carboxylated polystyrene in which a peptideGPIIb/IIIa receptor ligand, such as fibrinogen, is covalently bonded tothe bead surface by means of a covalent bond between the N-terminus ofthe peptide and an N-hydroxysuccinimide or carboxylate group on the beadsurface in a further embodiment, the kit additionally comprises aplatelet activator, such as a composition of ADP and PGE1.

EXAMPLE

The following examples are offered by way of illustration and withoutlimitation. Parts and percentages are by weight unless otherwiseindicated

The following examples and preparations are intended to illustrate theinvention but are not intended to limit its scope.

Dose response testing was performed with ADP (Chronolog) and PGE1(SIGMA) at 20 μM and 22 nM final concentrations respectively. ADP wasdiluted in Hepes/Saline, pH 7.4 buffer to a final concentration of 200μM prior to use on the aggregometer. PGE1 was diluted in Hepes/Saline,pH 7.4 buffer to a final concentration of 220 nM prior to use on theaggregometer. A P2Y12 receptor blocker was diluted in DMF to finalconcentrations of 1 mM, 2 mM and 5 mM.

Five microliters of the diluted P2Y12 compound were spiked into 5 mLwhole blood. Samples were inverted and incubated for one hour at roomtemperature. The whole blood baseline sample did not receive anyadditive. Once incubation was complete, whole blood samples were spun at1500 rpm for 15 minutes for platelet rich plasma (PRP) and 3500 rpm for15 minutes for platelet poor plasma (PPP). Platelet count was adjustedto approximately 250,000 μL for each sample using PPP.

For aggregometry, 450 μL of adjusted PRP was added to the glass cuvette.The blank sample contained 450 μL PPP and 50 μL Hepes/Saline buffer.Fifty microliters of a composition of 200 μM ADP and 220 nM PGE1 wasadded to each PRP sample and tested for ten minutes on the aggregometer.

Results

As illustrated in FIG. 3, the above reagents and system successfullydetects the extent of platelet aggregation from a blood sample treatedwith an P2Y12 antagonist. FIGS. 1 and 2 illustrates the mean response ofone individual and five individuals, respectively.

It is evident from the above results illustrated in FIG. 3 that asimple, rapid method is provided by the present invention for conductingan assay for platelet activity on samples that have been affected byexposure to a P2Y12 antagonist.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

The invention claimed is:
 1. A method for measuring inhibition ofplatelet aggregation by a P2Y12 antagonist, comprising the steps of: (a)providing a platelet containing blood sample from an individual treatedwith a P2Y12 antagonist; (b) contacting the platelet containing bloodsample with particles comprising an attached GPIIb/IIIa receptor ligand,adenosine diphosphate (ADP) and prostaglandin El (PGE1) under conditionssuitable for agglutination of said particles mediated by the platelet inthe blood sample, and (c) assessing the agglutination to determine thelevel of inhibition of platelet aggregation by the P2Y12 antagonist inthe individual, wherein the level of agglutination indicates whether theindividual has reduced ability to form platelet aggregation in responseto the P2Y12 antagonist treatment.
 2. The method of claim 1, wherein theP2Y12 antagonist is a thienopyridine.
 3. The method of claim 2, whereinthe thienopyridine is clopidogrel.
 4. The method of claim 1, wherein theplatelet containing blood sample is from an individual treated with aP2Y12 antagonist and aspirin.
 5. The method of claim 1, wherein theparticles comprise polystyrene or latex.
 6. The method of claim 1,wherein the particles comprise an infrared dye, the contact between theplatelet containing blood sample and the particles forms an assaymixture, and the agglutination of the particles mediated by the plateletis assessed by irradiating the assay mixture with a light in theinfrared spectrum, and assessing the transmission of infrared light fromthe assay mixture.
 7. The method of claim 1, wherein the GPIIb/IIIareceptor ligand comprises a substance selected from the group consistingof fibrinogen, monoclonal antibody 10E5, monoclonal antibody c7E3, vonWillebrand factor, fibronectin, vitronectin, a ligand that has anarginine-glycine-aspartic acid (RGD) sequence, a peptide that mimics theRGD sequence, and a peptidomimetic that mimics RGD sequence.
 8. Themethod of claim 1, wherein the GPIIb/IIIa receptor ligand is fibrinogen.9. The method of claim 1, which is conducted at a temperature rangingfrom 30° C. to 40° C., and the total time of the readings from the timeof the contact among the platelet containing blood sample, the particlescomprising an attached GPIIb/IIIa receptor ligand, ADP and PGE1 rangesfrom about 10 seconds to about 10 minutes.
 10. The method of claim 1,wherein the platelet containing blood sample is a whole blood sample ora plasma sample.
 11. The method of claim 10, wherein the plasma sampleis a platelet rich plasma (PRP) sample.
 12. The method of claim 1,wherein the particles, ADP and PGE1 are contained in an assay medium.13. The method of claim 12, wherein the particles, ADP and PGE1 arecontained in an assay medium on a disposable cartridge.
 14. The methodof claim 12, wherein the assay medium has an absorption peak at about800 nm.
 15. The method of claim 2, wherein the thienopyridine isticlopidine.